Abstract

Objective

To describe Japanese macaque encephalomyelitis (JME), a spontaneous inflammatory demyelinating disease occurring in the Oregon National Primate Research Center’s (ONPRC) colony of Japanese macaques (JM, Macaca fuscata).

Methods

JM with neurologic impairment were removed from the colony, evaluated and treated with supportive care. Animals were humanely euthanized and their central nervous system (CNS) examined.

Results

ONPRC’s JM colony was established in 1965 and no cases of JME occurred until 1986. Since 1986, 56 JM spontaneously developed a disease characterized clinically by paresis of one or more limbs, ataxia or ocular motor paresis. Most animals were humanely euthanized during their initial episode. Three recovered, later relapsed and were then euthanized. There was no gender predilection and the median age for disease was 4 years. Magnetic resonance imaging of eight cases of JME revealed multiple gadolinium enhancing T1-weighted hyperintensities in the white matter of the cerebral hemispheres, brainstem, cerebellum and cervical spinal cord. The CNS of monkeys with JME contained multifocal plaque-like demyelinated lesions of varying ages, including acute and chronic, active demyelinating lesions with macrophages and lymphocytic periventricular infiltrates, and chronic, inactive demyelinated lesions. A previously undescribed gamma-herpesvirus was cultured from acute JME white matter lesions. Cases of JME continue to affect 1–3% of the ONPRC colony per year.

Interpretation

JME is a unique spontaneous disease in a nonhuman primate that has similarities with multiple sclerosis (MS) and is associated with a novel simian herpesvirus. Elucidating the pathogenesis of JME may shed new light on MS and other human demyelinating diseases.

INTRODUCTION

Multiple sclerosis (MS) is a chronic inflammatory disease that affects the central nervous system (CNS). The disease typically follows a relapsing-remitting course and is associated with a variety of neurological deficits, such as paralysis, imbalance, ataxia, blindness and ocular motor paresis (reviewed in 1). Classically, the pathology of MS consists of discrete, multifocal areas of demyelination, associated with variable axonal loss in the white matter of the brain, spinal cord and optic nerves. More recently, lesions within the grey matter have been described. Lesions with active demyelination are associated with infiltrating lymphocytes and lipid-laden phagocytic macrophages. Although the cause of MS remains uncertain, there is considerable evidence indicating that multiple genetic loci and incompletely understood environmental exposures influence the risk of developing MS (reviewed in 2). One still unproven hypothesis is that a viral infection occurring in genetically susceptible individuals induces the disease.

The most widely studied model of MS is experimental autoimmune encephalomyelitis (EAE) (reviewed in 3). This paralytic illness can be induced in a variety of mammalian species by immunization with whole myelin or specific myelin proteins, most notably myelin basic protein (MBP), myelin oligodendrocyte glycoprotein and proteolipid protein, or peptides from these proteins4–6. Depending on the species used and method of disease induction, EAE can follow a chronic relapsing course and be associated with multifocal areas of demyelination and thus mimic MS pathologically and clinically7. There are also mouse models of virally induced demyelinating diseases, most notably Theiler’s murine encephalomyelitis virus (TMEV)8–10. TMEV-induced demyelinating disease (TMEV-IDD) is induced by injecting neurotropic strains of the virus into genetically susceptible mice11. Depending upon the strain of TMEV, infection can result in either acute encephalitis or a chronic immune-mediated multifocal demyelinating disease. While EAE can be induced in rhesus and common marmoset monkeys, a virally induced model of MS has not been described in nonhuman primates (NHP). Availability of a demyelinating disease associated with a virus in an NHP might prove relevant to understanding the pathogenesis of MS.

Here, we report the spontaneous occurrence of an inflammatory demyelinating disease in a colony of Japanese macaques (JM, Macaca fuscata) at the Oregon National Primate Research Center (ONPRC). The disease, Japanese macaque encephalomyelitis (JME), has clinical and pathologic similarities with MS. Moreover, we report the isolation of a previously undescribed simian herpesvirus isolated from acute JME lesions. This is the first description of a spontaneously occurring MS-like disease in a NHP and has the potential to offer new insights into the pathogenesis of MS.

MATERIAL AND METHODS

Animals

All animal procedures were approved by the ONPRC Institutional Animal Care and Use Committee. The ONPRC is an Association for Assessment and Accreditation of Laboratory Animal Care International accredited research facility and conforms to National Institutes of Health guidelines on the ethical use of animals in research. The JM colony is maintained in a two-acre corral and members of the colony are observed daily for the presence of sick or injured animals. Per institutional protocol, members of the colony developing neurologic dysfunction were removed from the corral, placed in cages, evaluated and provided supportive care. Most animals developing JME were humanely euthanized per ONPRC protocol. Three animals recovered from their initial episode, were returned to the corral, but later relapsed and then were humanely euthanized.

For quantitation of olig2-immunolabeled cells, three adjacent sections were analyzed at 40× following double labeling with anti-MBP and anti-olig2 antibodies. Three regions of interest (ROI) were identified using MBP labeling: the lesion, where MBP immunoreactivity was mostly absent; the border, where there was a mixture of intact myelin and damaged myelin; adjacent unaffected white matter. The number of olig2+ cells in three random microscopic fields within each ROI in each adjacent section was then counted and averaged. The mean differences between ROIs were assessed using Student’s t test.

Magnetic resonance imaging

A 4 year 240 day old female JM exhibiting signs of JME was transported to Oregon Health & Science University Hospital and positioned in a General Electric 1.5 Tesa (T) Signa MRI instrument and T2 weighted and pre- and post-gadolinium T1 weighted images were obtained from the brain and cervical cord. All other MRIs were performed on a 3 Tesa Siemens Trio whole-body MRI instrument using a quadrature transmit/receive extremity radiofrequency coil. Sixty sagittally oriented 2D turbo spin echo acquisitions with proton density (TE17/TR9000/ETL9/NEX1) contrast were collected with a 1 mm slice thickness and 256×256 matrix over a 160mm2 field-of-view (FOV). Multislice 2D turbo spin echo MRI acquisitions with proton density (TE13/TR9000/ETL9/NEX2) and T2-weighted (TE92/TR9000ETL9/NEX2) contrast were collected in an axial orientation angulated parallel to the imaginary line connecting the anterior and posterior commissures; 66 1 mm slices were collected using a 320×240 matrix over a 160mm×120mm field-of-view (FOV). Transaxial 3D T1-weighted Magnetization Prepared Rapid Acquisition Gradient Echo (MPRAGE); TE3.5/TR2500/TI900/FA8) data sets were acquired before and 30 minutes after gadoteridol administration with a 192×144×96 matrix over a 130mm×98mm×76mm field-of-view (FOV). Gadoteridol was administered in situ at a dose of 0.2 mmol/kg using an infusion pump.

Isolation of a herpesvirus from an acute lesion

A spinal lesion from a JM (#17792) with JME was obtained at necropsy, homogenized and placed over primary rhesus macaque fibroblasts for isolation and analysis of a potential infectious agent as described12. Preliminary molecular characterization to identify the herpesvirus was conducted as described12. Using the same technique, attempts were made to isolate viruses from other acute JME lesions and normal white matter from JM with and without JME.

RESULTS

Japanese macaque encephalomyelitis

The ONPRC has housed an outbred colony of JM since 1965. No cases of unexplained neurologic disease were observed in the colony prior to 1986. Since 1986, 56 JM spontaneously developed JME (Figure 1). Currently, 1–3% of the colony develops JME each year and the disease affects males and females equally. Except for the burst of cases in August 1987, there is no apparent seasonal predilection and cases have occurred every year since 1986 except in 1993 and 2004. Table 1 provides details of the clinical features of the disease on the 56 cases. The median age of affected animals was 4 years 109 days (range, 97 days-26 years 50 days). The majority of the animals had paresis or paralysis of one or more limbs and ataxia. Ocular paresis occurred in 10 cases. Seven animals exhibited other clinical manifestations, including abnormal species-specific behaviors and head tilt accompanied with body tremors. Typically, the onset of clinical disease was acute and progressed rapidly with the median time between presentation of disease to humane euthanasia being 6 days (range, 0 days-121 days). In most cases, euthanasia was required because neurologic impairment made it impossible to return the monkeys safely to the colony. Three animals (#12068, 21255 and 16749) recovered from their initial episode and were returned to the corral, but were later euthanized when their JME recurred 101 days-8 years later.

CSF was collected on 40 affected animals. The mean CSF white blood cell (WBC) count was 185 cells/mm3 with a range from 0 to 2,710. WBC differential was obtained in 13 cases and in all but one the cells consisted of both lymphocytes and neutrophils. The mean total protein was 52 mg/dL (range, 0–195 mg/dL). Glucose values were normal and attempts to isolate bacteria from CSF were negative. Hematological and biochemical examinations of blood revealed no consistent abnormalities. CSF was not examined for oligoclonal bands or IgG index.

MRI abnormalities

MRI scans were performed on eight animals with JME and MRI scans from three cases are shown in Figure 2. A brain and cervical MRI scan was performed on JM #19384 11 days after presentation with clinical signs of JME (Figure 2A–C). Post-gadolinium T1-weighted images revealed multiple areas of focal contrast enhancement in the white matter of the cerebral hemispheres, cerebellum, brainstem and cervical cord. JM #26174 underwent MRI 4 days after presentation with acute JME signs (Figure 2D–I). The T2-weighted axial images in D-F showed hyperintense areas in the upper cervical cord, cerebellum and cerebrum. The axial images of G-I show intense focal contrast enhancement in the upper cervical cord, cerebellum and genu of the corpus callosum. JM #13221 underwent MRI 3 days after presentation with acute JME symptoms (Figure 2J–L). T2-weighted axial images revealed a large hyperintense area in the left cerebral peduncle (Figure 2J). The same area had a hypointense appearance in the T1-weighted MPRAGE image (Figure 2K). Punctate gadolinium enhancement was apparent in this same peduncle region in the post-contrast T1-weighted image (Figure 2L). The MRI abnormalities displayed here are similar to those of the other five cases of JME that underwent MRI, consistent with active neuroinflammation and similar to the MRI findings of acute lesions in MS.

Neuropathology

Complete necropsies were performed on 54/56 cases of JME. No monkeys had evidence of a systemic illness or acute disease outside of the CNS. In all cases, neuropathologic examination of the brain and cervical spinal cord revealed a multifocal demyelinating encephalomyelitis. In most cases, multiple, dull, yellow-white to gray-tan, well-delineated plaques that ranged from 0.2 to 1.0 cm in greatest dimension were apparent on gross inspection of post-fixed coronal brain and spinal cord slices. Gross lesions were most frequent in the cerebellar white matter, brachium pontis, pyramids and the white matter of the cervical spinal cord. A typical distribution of plaque-like lesions in the cerebellum and spinal cord was found in animal #19384 that underwent MRI (Figure 3A). Histologically, a majority of lesions seen in all JME cases had morphologic features consistent with the chronic active plaques described in MS13–17. Plaques were typically centered on venules or small veins. The chronic active plaques (Figure 3B) were dominated centrally by macrophages engorged with myelin debris intermingled with astrocytes. Infiltrating macrophages and lymphocytes dominated in peripheral portions of plaques with focal concentrations of lymphocytes forming perivascular cuffs. Areas of necrosis and microcystic spaces were occasionally observed in the central portion of chronic-active lesions. Variations in lesion age were observed in some animals. Early lesions were infrequent and characterized by perivenous lymphocytic cuffs that frequently tracked perforating venules from piaarachnoid surfaces into adjacent white matter. The adjacent myelin was vesiculated and sparsely infiltrated with lymphocytes and macrophages. Rarely, small focal hemorrhages were encountered in the edematous myelin. Chronic inactive plaques composed principally of astrocytes and foamy macrophages were seen in many of the animals (Figure 3C). Lymphocytes were largely absent in these lesions and silver staining revealed decreased numbers of axons with evidence of acute axotomies (Figure 3D and E). Regardless of apparent age, lesions were exclusively located in white matter in all animals. Apart from rare, small, perivascular collections of lymphocytes, lesions were not seen in grey matter or the meninges. Transmission electron microscopy (EM) was performed on a limited number of lesions and these disclosed degenerating axons, demyelinated axons and occasional thinly myelinated axons, suggesting remyelination (data not shown).

Immunostaining was performed on two lesions localized by MRI in animal #13221. The first lesion was analyzed by immunostaining for alterations in MBP, NF, and olig2 immunoreactivity, and a second for infiltration of macrophages and T cells. MBP and NF labeling demonstrated areas with axon and myelin debris (defined as the demyelinated lesion) adjacent to border areas showing a mixture of intact and damaged fibers, which was, in turn, adjacent to unaffected white matter (Figure 4A and B). Staining for the presence of CD68 in a lesion located in the cerebellum revealed numerous CD68+ macrophages and perivascular CD3+ T cells (Figure 4C). Oligodendrocytes were quantified by enumerating olig2-labeled cells within the lesion shown in Figure A and B. There was nearly complete absence of olig2 immunoreactivity within the lesion and a dramatic reduction in olig2+ cells at the lesion border compared to adjacent unaffected white matter (Figure 4D and E). The lesions of JME thus are multifocal, contain T cell and macrophage infiltrates and reveal demyelination, loss of oligodendrocytes, limited remyelination, and axonal degeneration.

Isolation of novel gamma-2 herpesvirus from encephalomyelitis lesions

As viral infection has been associated with inflammatory demyelination in mice, we investigated whether a virus might be present in acute lesions of animals with JME. Explant cultures of lesioned spinal cord tissue versus normal neuronal tissue from JM #17792 layered over primary rhesus fibroblasts yielded an infectious agent that was identified by transmission EM as a herpesvirus (data not shown). The virus isolate was subsequently passaged and viral DNA purified from cell-free virions for degenerate PCR amplification over a highly conserved region of the viral DNA polymerase to identify the virus. Preliminary DNA sequence analysis revealed the virus was most similar to rhesus macaque rhadinovirus (RRV). The complete sequence of the viral genome was determined and compared to simian and human herpesvirus genomes representing alpha-herpesviruses, beta-herpesviruses and gamma-1 and -2 herpesviruses. By this analysis, the JM herpesvirus, referred to as JM rhadinovirus (JMRV) is a gamma-2 herpesvirus most closely related to RRV, with 89.5% nucleotide sequence identity and secondarily related to human Kaposi sarcoma herpesvirus (KSHV) with 47.9% nucleotide sequence identity (Table 2). JMRV has subsequently been isolated from active CNS lesions of five other JM with JME, but has not been isolated from normal white matter from JM with or without JME.

DISCUSSION

JME appeared spontaneously in JM at the ONPRC 21 years after the colony was established. The initial case of JME occurred in 1986 and since then the disease typically has affected 1–3% of the colony each year, with two exceptions. JME usually occurs in young adult animals, but the disease can present in juvenile or aged animals, and shows no preference for either gender. Clinically, JME causes paralysis, ataxia and ocular motor paresis. While most animals failed to recover sufficiently to be safely returned to the colony, three monkeys recovered, were returned to the colony and then relapsed one or more years later. MRI of eight animals revealed changes similar to acute MS. Pathologically, JME causes multifocal areas of demyelination of varying acuity with loss of oligodendrocytes and variable axonal loss in the white matter of the cerebrum, cerebellum, brainstem and spinal cord associated with macrophages and lymphocytic infiltrates. Chronic inactive demyelinated lesions also occur, suggesting that asymptomatic lesions may occur before clinical JME. JME is the first naturally occurring inflammatory demyelinating disease in an NHP.

JME has clinical, MRI and pathologic similarities to MS. These include similar clinical manifestations, a relapsing course in monkeys that recovered sufficiently to be returned to the colony, MRI abnormalities like those of acute MS lesions and similar neuropathologic abnormalities, including plaque-like demyelinating lesions associated with lymphocytes and macrophages, oligodendrocyte depletion, limited remyelination and variable axonal loss. However, there are some features of JME that differ from MS. These include CSF containing neutrophils as well as lymphocytes in most cases and necrosis and hemorrhage as part of the pathologic continuum. These differences may represent species-specific differences in inflammatory and tissue responses. It is worth noting that EAE in NHP often has neutrophils in the CSF and necrosis and hemorrhage as part of the neuropathology, which differs from EAE in rodents18. Overall the clinical, MRI and neuropathologic features of JME have more similarities than differences with MS and JME is clearly an inflammatory demyelinating disease. Further investigations are needed to determine the full extent of similarities of JME with MS.

There are several features of JME that makes this an extremely appealing model for MS. First, the disease occurs spontaneously, which makes it distinct from current models of MS that require artificial manipulation to induce disease. Second, it affects a small percentage of animals in the colony, approximately 1–3% each year, suggesting there may be a genetic susceptibility to the disease as there is in MS. The ONPRC began tracing the pedigrees of the JM troop in 2000 utilizing specific microsatellite markers. Interestingly, animals developing JME since 2000 come from distinct matrilines from the original troop, supporting the idea that host genetic factors play a significant part in susceptibility to disease (unpublished data). Determining the genetic factors that coincide with disease and predispose animals to develop JME is currently being explored on archived tissue and new cases. Third, like MS, JME is an inflammatory disease, suggesting either an autoimmune disease or a viral infection. An autoimmune pathogenesis of MS is commonly proposed but definitive evidence of this remains lacking. Auto-reactive T cells and anti-myelin antibodies have been proposed as being critical to the immunopathogenesis of MS19–21. We are currently assessing new cases of JME for anti-myelin T cell responses and antibodies against myelin antigens.

Finally, based on epidemiologically studies and the histopathology, a viral etiology for MS has long been suspected. Multiple candidate viruses have been proposed but none has been convincingly associated with the disease. Here, we report the isolation of a previously unknown herpesvirus, JMRV, isolated from acute JME lesions. We have been unable to isolate JMRV from normal appearing white matter of JM with and without JME. Complete DNA sequencing reveals this to be a rhadinovirus with significant sequence homologies with RRV and human KSHV. Reagents are being generated against this virus to evaluate the potential role of JMRV in JME. This is of particular interest since human Epstein-Barr virus and human herpesvirus 6 are two herpesviruses that have been implicated as playing a role in the pathogenesis of MS22–30. Demonstrating that a simian rhadinovirus induces JME will provide a new class of herpesvirus that would warrant investigation in MS.

In summary, JME is a unique spontaneous demyelinating disease in an NHP. Preliminary results suggest that the disease occurs in genetically predisposed monkeys and is associated with a novel simian herpesvirus. Further investigation of the pathogenesis of JME may provide new insights into the pathogenesis of MS.

ACKNOWLEDGEMENTS

The authors thank Ms. Lori Boshears and Mr. Andrew Townsend for assistance with the manuscript and figures, and members of the Division of Animal Resources for their diligence and care of the animals. This work was supported by the National Institutes of Health (NIH) Grant P51RR00163, the Research Enrichment Award Program of the Department of Veterans Affairs Biomedical Laboratory Research and Development, the OHSU Multiple Sclerosis Center, the United States Department of Defense (W81XWH-09-1-0276), OHSU/Vertex SRA-05-07-01 and NIH Grant R01-EB007258.